An overview of calf diarrhea - infectious etiology, diagnosis, and intervention

Cho, Yong-il; Yoon, Kyoung-Jin

Real-time PCR (as known as quantitative PCR or qPCR) is a method that is capable of not only amplifying the target sequence but also quantifying the amount of target with great sensitivity and high throughput [142] . There are three types of real-time PCR methods commonly used for diagnostic purposes: TaqMan, molecular beacon, and SYBR Green real-time PCR. TaqMan real-time PCR involves a oligonucleotide probe labeled with two types of fluorophores (i.e., a report dye and quencher dye) in addition to a primer pair [31] . The reporter dye is located on the 5´-end of the probe and the quencher is attached to the 3´-end. After denaturation of the DNA template, the primers and probe bind to each strand of the template. Extended primers remove the TaqMan probe from the template DNA, and the reporter dye is thus separated from the quencher dye. Emission from the reporter dye (e.g., fluorescence energy) can be detected spectrophotometrically. All real-time PCR steps are conducted in a closed tube system; hence, the opportunity for contamination can be minimized. The assay provides high specificity due to detection of probe signal based on primer extension. Real-time PCR using a molecular beacon probe is similar to TaqMan real-time PCR. However, the beacon probes form a hair-shape structure since the probe sequence is placed between the "arm" sequences and produce bright fluorescence when bound to their target template [80] . The principle of SYBR Green real-time PCR is based on SYBR Green dye binding to double-stranded DNA that will produces light when excited. SYBR Green assays are cheaper than TaqMan real-time PCR techniques. However, the dye binds to any double-stranded DNA molecule. Therefore, SYBR Green real-time PCR requires a melting-curve analysis to determine whether the amplification curve is produced by the intended target or other factors such as primer dimers or non-specific amplicons [59] .